Cathepsin D as a prognostic indicator for node-negative breast cancer patients using both immunoassays and enzymatic assays.

This is a retrospective study on 162 node-negative patients, with both biochemical and clinical factors being measured for determination of prognostic markers. Steroid receptors were measured on all tumors, while tumor size, histological grade, ploidy status, and cell cycle kinetics indicators could not be found or measured on 25 or less of the patient group. The primary focus of this study was the measurement of cathepsin D, analyzed by two different procedures, and 161 of the 162 patients had at least one value. The antigenic assay was performed using the US-CIS kit, and it was sensitive and reproducible. A biochemical assay using the enzymatic activity of cathepsin D was developed, and it gave proportional values, compared to the antigenic assay values (r2 = 0.79). Our results indicated that the mean antigenic levels were 20% higher than the biochemical assay levels (P = 0.001). High levels of cathepsin D by the antigenic assay predicted poor relapse-free (P = 0.0001) and overall (P = 0.0004) survival. High levels of cathepsin D by the biochemical assay also predicted poor relapse-free (P = 0.031) and overall (P = 0.0013) survival. The cathepsin D values were still useful as predictors of outcome after multivariate analysis. Several other factors, such as grade and S phase, were useful as additional prognostic indicators. In conclusion, cathepsin D is the most useful marker in node-negative patients, and the analysis can be performed by both a biochemical and an antigenic assay.     

Prognostic significance of DNA-ploidy in a series of 690 primary breast cancer patients

Flow cytometric DNA-ploidy measurements were performed on paraffin-embedded and fresh tumor specimens from 690 patients with Stage I-III breast cancer. The conventional classification of DNA-ploidy (diploid versus aneuploid) was compared with a division of tumor ploidy into 5 classes based on DNA index (DI) range. The DI-classification showed a better correlation with tumor size and TNM stage than the conventional classification. Aneuploidy was associated with an impaired survival and distant relapse-free survival (p = 0.02) but the DI-classification improved the discrimination between different prognostic groups of patients. In general, this indicated a more aggressive phenotype for tumors evolved via polyploidization. Hyper-tetraploidy (DI greater than 2.10) indicated a very poor prognosis in pre-menopausal patients. No prognostic effect of aneuploidy and DI-class was found in node-negative and TI patients. Cox multivariate regression analysis showed that aneuploidy was an additional prognostic factor to nodal status (I less than or equal to N less than or equal to 3, N greater than 3 vs. N = 0) and tumor size (T2-4 vs. TI) for overall and distant relapse-free survival. Subdivision according to DI-class did not improve the prognostic power of DNA-ploidy due to stronger correlations with established prognostic factors.     

Preoperative values of CA 15-3 and CEA as prognostic factors in breast cancer: a multivariate analysis.

The role of circulating tumor markers in providing prognostic information has not been widely studied. In the current study, serum levels of the carbohydrate antigen 15-3 (CA 15-3) and carcinoembryonic antigen (CEA) were determined preoperatively in 364 breast cancer patients with no clinical signs of metastasis. The prognostic relevance of these markers for recurrence (175/364) and death of disease (104/175) was determined by Cox multivariate analysis, including the comparison with classical prognostic factors. High levels of both tumor markers were associated with aneuploid tumors with high S-phase fraction and high ornithine decarboxylase activity. CA 15-3 was highly associated with the number of positive lymph nodes and peritumoral lymphatic or blood vessel invasion. No significant associations were found between CEA or CA 15-3 levels and histologic grade, necrosis and steroid receptor status. In univariate analysis, preoperative values, using optimum cutoff values of CA 15-3 (40 U/ml) and CEA (6 ng/ml), were statistically significant for relapse-free survival and overall survival. In multivariate analysis, only node status, DNA ploidy and ornithine decarboxylase activity were independent predictors for relapse-free survival; the estrogen receptor status was a predictor of overall survival. In node-negative patients, ornithine decarboxylase activity was the only factor selected for relapse-free survival. In node-positive patients, the number of lymph nodes and DNA ploidy were the only variables selected for relapse-free survival or overall survival. Estrogen receptor and ornithine decarboxylase activity were excluded for relapse-free survival, but were significant prognostic factors for overall survival.     

Tumour DNA ploidy as an independent prognostic factor in breast cancer

We determined nuclear DNA content from 308 archival paraffin-embedded malignant breast tumours and evaluated the survival of the patients by univariate and multivariate statistical analyses. The overall 8-year survival rate of stage I-III breast cancer patients was 74.3% in DNA-diploid and 51.2% in DNA-aneuploid tumours (P less than 0.0001). DNA ploidy had prognostic significance in both node-negative and node-positive breast cancer, primarily in cases with steroid receptor-positive tumours. In a Cox multivariate analysis DNA ploidy (P = 0.001), primary tumour size (P = 0.0007), nodal status (P = 0.04) and the content of progesterone receptors (P = 0.0008) emerged as significant independent prognostic factors, whereas oestrogen receptor status, age and menopausal status of the patients had no significant independent prognostic value. If the histological grade of ductal carcinomas was also included in the Cox model, both grade and DNA ploidy had independent prognostic effect. In conclusion, our results indicate that the analysis of DNA ploidy is a useful adjunct in the assessment of prognosis for breast cancer patients.     

Node-negative breast cancer: prognostic subgroups defined by tumor size and flow cytometry

Adjuvant systemic therapy for women with node-negative breast cancer is most easily justified for those patients at highest risk of relapse. We have examined the impact of tumor size, histologic grade, estrogen receptor (ER) status, tumor ploidy, and S-phase fraction (SPF) on relapse-free survival (RFS) for 169 patients with node-negative breast cancer in order to identify groups of patients at high and low risk of relapse. Patients with small tumors (less than or equal to 1.0 cm) had a significantly better RFS than those with larger tumors (P = .005), with 96% remaining relapse-free at 5 years. Patients with tumors less than or equal to 1.0 cm were thus excluded from analysis when attempting to define a group with a poor prognosis. Within the group of patients with tumors greater than 1.0 cm, tumor ploidy (P = .63), ER status (P = .3), or progesterone receptor (PgR) status (P = .24) did not predict for RFS. Patients with grade 1 or 2 infiltrating ductal tumors had a significantly better prognosis than those with grade 3 tumors (P = .04). The prognostic factor that gave the widest separation between subgroups, however, was SPF. Patients whose tumors were greater than 1.0 cm with an SPF less than or equal to 10% had a 5-year RFS of 78% compared with a 5-year RFS of 52% for those with an SPF greater than 10% (P = .006). We have combined tumor size and SPF to identify three prognostic groups: (1) tumor less than or equal to 1.0 cm, 5-year RFS 96%; (2) tumor greater than 1.0 cm plus SPF less than or equal to 10%, 5-year RFS 78%; 3) tumor greater than 1.0 cm plus SPF greater than 10%, 5-year RFS 52%. These prognostic groupings may help identify patients most suitable for adjuvant therapy.     

The prognostic significance of tumor ploidy and pathology in adenocarcinoma of the esophagogastric junction

Tumor DNA content has been advocated to be an important prognostic indicator in human malignancies. Paraffin-embedded specimens of 75 resected adenocarcinomas (AC) of the esophagogastric junction were studied by flow cytometric DNA analysis to determine whether tumor ploidy was a significant prognostic variable independent of stage and histologic grade of the tumor. Eighty-one percent of the tumors were aneuploid. More patients with aneuploid tumors had lymph node metastases than patients with diploid tumors (P = 0.007). Patients with aneuploid tumors had poorer 18-month disease-free and overall survival than patients with diploid tumors. Cox regression analysis demonstrated that the most important prognostic variables for predicting overall survival were lymph node status, depth of wall invasion, and tumor differentiation. Tumor ploidy was not an independent prognostic variable in predicting recurrent disease or death from AC of the esophagogastric junction. Tumor DNA content is valuable, however, as a marker for patients at increased risk of lymph node metastases, early recurrence, and poorer survival.     

c-myc amplification is a better prognostic factor than HER2/neu amplification in primary breast cancer.

Amplification of the c-myc and HER2/neu genes was found in 20 and 23%, respectively, of primary breast cancer tissues derived from 282 patients (median follow-up, 74 months). c-myc amplification was observed more frequently in larger tumors (P = 0.01) and in lymph node-positive patients (P = 0.01) but was not associated with age, menopausal status, or with differentiation grade or steroid receptor status. c-myc amplification was strongly negatively correlated with HER2/neu amplification (P less than 0.001). In univariate analysis, amplification of c-myc proved to be a significant predictor of reduced relapse-free and overall survival (for both, P less than 0.001). In multivariate analysis for relapse-free survival, c-myc amplification significantly (P = 0.001) added to the prognostic power of tumor size (P less than 0.001), lymph node status (P less than 0.001), and estrogen receptor status (P = 0.003), with the highest relative failure rate (1.8) after lymph node status (2.2). In this pilot study, c-myc amplification was predictive for outcome, especially among patients with node-negative disease or steroid receptor-positive tumors; 51 and 46% differences in actuarial 5-year recurrence rates when compared to patients with tumors with normal c-myc gene copy numbers, respectively. HER2/neu amplification was not associated with relapse-free survival but weakly with shorter overall survival in univariate analysis (P = 0.035). Only in the relatively small subgroup of steroid receptor-negative tumors, HER2/neu amplification may identify those patients with an increased risk of death. In conclusion, amplification of c-myc is an independent powerful prognosticator, particularly in node-negative and steroid receptor-positive breast cancer, whereas HER2/neu amplification may be of limited prognostic value, only in steroid receptor-negative disease.     

S-phase fraction and DNA ploidy in 633 T1T2 breast cancers: a standardized flow cytometric study.

The lack of a standardized methodology for quantifying DNA ploidy and S-phase fraction (SPF) by flow cytometry is hindering routine use of these markers in breast cancer management. In a retrospective clinical multicenter study, we validated a standardized flow cytometry protocol. We tested 633 frozen T(1)T(2), N(0)N(1), M(0) breast tumors obtained in four institutions. Cell preparation was standardized, and precise rules for data interpretation were followed. Three SPF classes were defined on the basis of tertiles after adjustment for ploidy. DNA aneuploidy was observed in 61.0% of cases. No significant difference was observed among centers. Aneuploidy and high SPF were associated with large tumor size, node involvement, high histological grade, and hormone receptor negativity. In the overall population (median follow-up, 69 months), patients with medium and high SPF values had shorter disease-free survival (DFS) than those with low SPF values (P < 0.0001). Ploidy had no significant influence. By Cox analysis, SPF, pN, and estrogen receptor status were independent predictors of DFS (P = 0.0002, P = 0.001, and P = 0.05). In node-negative patients, SPF was the only predictor of DFS (P = 0.01), whereas in node-positive patients, the risk of relapse increased with both high SPF (P = 0.003) and estrogen receptor negativity (P = 0.004). Low SPF values distinguished grade II tumors with a particularly good outcome. Our results strongly support the use of SPF in multicenter studies and clinical trials and suggest that node-negative patients with slowly proliferating tumors do not require systemic adjuvant therapy.     

Premenopausal node-negative breast cancer: May adjuvant chemotherapy be indicated by the analysis of nuclear DNA dynamics?

The management of premenopausal node-negative breast cancer patients is discussed controversially. Accurate cellular as well as biochemical markers are essential for this cancer group to identify high risk patients needing adjuvant chemotherapy. In the present study, flow cytometric DNA analysis (DNA-ploidy status, DNA-index, S-phase fraction, S+(G₂+M)-phase fraction) and clinico-pathological variables (clinical stage, tumor size, receptor status, age, histological type and grade) as prognostic factors were determined on paraffin-embedded tumors to predict overall survival (OS) and disease-free survival (DFS). Median observation time was 6.1 years (n = 57). S+(G₂+M)-phase fraction was the only flow cytometric DNA predictor of overall survival in the univariate analysis (log-rank test): As compared to the patients with lower S+(G₂+M)-phase fraction ( 9.3%), patients with S+(G₂+M)-phase fraction greater than 9.3% had shorter survival (P = 0.039). Of all the clinico-pathological parameters analyzed (univariate analysis), the survival time was found to be longer when estrogen- and/or progesterone-receptor status was positive (overall survival: P = 0.039; disease-free survival: P = 0.017) and the histological grade was low (overall survival: I + II vs III: P = 0.024; I vs II vs III: P = 0.046). In the multivariate analysis, receptor status was the strongest predictor for overall and disease-free survival. These results suggest that S+(G₂+M)-phase fraction in premenopausal node-negative breast cancer could be an additional valuable prognostic factor to classify high risk breast cancer patients needing adjuvant chemotherapy.     

Prognostic impact of cathepsin D and c-erbB-2 oncoprotein in a subgroup of node-negative breast cancer patients with low histological grade tumors

Some node-negative breast cancer patients, with initially good prognosis, relapse from their cancer and are poorly identified. In the present study, based on prospective data of 197 tumors, we measured cathepsin D (cath D, n=197), pS2 protein (n=125), c-erbB-2 oncoprotein (n=100) and epidermal growth factor receptor (EGF-R, n=99) to better define the risk of relapse of node-negative patients in comparison with that defined by the clinical and histological factors. The median follow-up in surviving patients was 75 months. Univariate analysis indicated that patients with histological grade III tumors (the Scarff, Bloom and Richardson classification) had a much poorer prognosis than those with histological grade I or II tumors (P=0.0027 for relapse-free survival and P=0.0156 for overall survival). When the population of node-negative patients was divided by tertiles, high cath D levels showed a significant association with an early relapse (P=0.0316). Using cut-off values, patients with high cath D (> or =25 pmol/mg protein) or c-erbB-2 oncoprotein (> or =4 Human Neu Unit/microg protein) levels, had a significant worse relapse-free survival (P=0.0147 and 0.0417, respectively). No prognostic information was supported by pS2 protein or EGF-R measurements. In multivariate analysis, histological grade, cath D and c-erbB-2 oncoprotein remained independent predictors of recurrence (P=0.005, 0.0361 and 0.0321, respectively). By combining low levels of cath D and c-erbB-2 oncoprotein in histological grade I or II tumors, we identified a subgroup of patients with a 100% relapse-free survival probability at 6 years of follow-up. Moreover, the subgroup of patients with histological grade I or II tumors and high values of both cath D and c-erbB-2 oncoprotein showed a prognosis as poor as the subgroup defined by histological grade III alone, respectively 66% and 70% relapse-free survival at 6 years of follow-up. In conclusion, the combination of conventional prognostic factor (histological grade) and biochemical factors (cath D and c-erbB-2 oncoprotein) enabled us to identify, in this preliminary study, a subgroup of patients having an increased risk of relapse in a group (node-negative patients with low histological grade tumors) considered as good prognosis.     

Flow cytometric analysis of nuclear DNA content in ovarian tumors. Association of ploidy with tumor type, histologic grade, and clinical stage.

The authors undertook a prospective, flow cytometric study of nuclear DNA ploidy in 140 fresh ovarian tumors. There were 43 benign tumors, 27 borderline tumors, and 70 malignant tumors. Results of DNA ploidy analysis were compared to age at diagnosis, menopausal status, tumor size, histologic type, grade, and International Federation of Gynecology and Obstetrics (FIGO) stage. Although the majority of benign tumors were diploid, 19% were aneuploid. Among the benign tumors, DNA ploidy was significantly associated with tumor type and tumor size. All borderline tumors were diploid. Of the 70 malignant tumors, 64% were aneuploid. In the malignant tumors, DNA aneuploidy had significant univariate associations with histologic type, grade, and FIGO stage. By multivariate analysis, DNA aneuploidy remained significantly associated with stage and grade, both known predictors of survival in ovarian cancer. These results indicate that DNA ploidy varies with the aggressive potential of an ovarian cancer and may, at the time of initial diagnosis, provide additional information about tumor prognosis.     

Prognostic significance of DNA ploidy, S-phase fraction, and P-glycoprotein expression in colorectal cancer

BACKGROUND AND OBJECTIVES: Parameters that allow prediction of the disease course in colorectal cancer would aid the development of improved treatment strategies. For this reason, we evaluated the prognostic value of flow cytometric DNA ploidy and S-phase fraction (SPF) and P-glycoprotein (Pgp) expression in this type of tumor. METHODS: The prognostic significance of DNA ploidy, SPF, and Pgp expression on paraffin-embedded sections from 107 patients with colorectal carcinoma was determined. The mean follow-up was 36.6 months (range = 3-72 months). DNA ploidy and SPF were evaluated by flow cytometry and Pgp by immunohistochemistry using monoclonal antibody C219. The Cox regression model was used to adjust for several clinical and pathologic covariates. RESULTS: Of the 107 carcinomas examined, 44 (41.1%) were classified as DNA diploid and 63 (58.9%) as DNA aneuploid. DNA ploidy pattern was significantly related to tumor site (P = 0.010), tumor stage (P = 0.016), and vascular invasion (P = 0.015) but not to other clinicopathologic variables. Patients with DNA diploid tumors showed a better survival rate than did those with aneuploid tumors. After stage IV disease was excluded, patients with diploid tumors also presented a better disease-free and overall survival than did patients with aneuploid tumors. Mean SPF of the whole series was 13.5% (median = 11.3%, range = 1.4%-29.9%). Aneuploid tumors had a higher median SPF than did diploid tumors (17 vs. 6.2; P = 0.0001). SPF was only related significantly with tumor location (P = 0.026). In the multivariate analysis, SPF was a significant independent prognostic factor for overall survival (P = 0.01). When stage IV was excluded, SPF was also an independent prognostic variable for both disease-free (P = 0. 02) and overall (P = 0.01) survival. Of 107 tumors, 61 (57%) were positive for Pgp expression, but no relation was found between this and other clinicopathologic parameters. Pgp expression had no influence on survival. CONCLUSIONS: Our results suggest that flow cytometric DNA ploidy and SPF are significant and independent prognostic factors in patients with colorectal carcinoma, whereas Pgp expression is not.     

DNA flow cytometry and prognostic factors in 1331 frozen breast cancer specimens

Breast cancer proliferative capacity as determined by the DNA thymidine labeling index, along with estrogen and progesterone receptor status, is highly predictive for risk of relapse and overall survival. Recently, DNA ploidy and proliferative capacity (S-phase fraction [SPF]) as determined by flow cytometry have also shown significant prognostic value. The authors have developed a technique which allows a 50 to 100 mg aliquot of the same frozen breast tumor specimen routinely employed in steroid receptor assays, to be assayed for both DNA ploidy and SPF by flow cytometry. Of the 1331 tumors examined, DNA histograms were evaluable for ploidy in 89% (1184) of specimens examined; 57% of these were aneuploid. Adapting a trapezoidal model to estimate SPF in both diploid and aneuploid tumors, the authors found 81% (1084) to be evaluable for SPF, with a median SPF of 5.8% for the entire population. The median SPF was significantly lower in diploid tumors (2.6%) than in aneuploid tumors (10.3%, P less than 0.0001). Both aneuploidy and high SPF were strongly associated with absence of steroid receptors. Aneuploid tumors showed more striking differences in the frequency of high S-phase values with respect to receptor status and age or menopausal status, whereas diploid but not aneuploid tumors showed lower SPF in node-negative versus node-positive patients. Because it is particularly important to identify the high-risk minority of node-negative patients, the authors examined the node-negative group separately. High SPF subgroups appeared in each category of receptor status and age or menopausal status within the node-negative group, suggesting that SPF will be an independent prognostic factor. With the DNA flow cytometric methods used here, it is now practical to determine ploidy and SPF for nearly every breast cancer patient. These factors, which show associations with established prognostic factors, such as receptor status can now be fully evaluated for their prognostic significance in broad patient populations.     

Breast carcinoma growth rate described by mammographic doubling time and S-phase fraction. Correlations to clinical and histopathologic factors in a screened population.

BACKGROUND. In a retrospective study, correlations among mammographic doubling times (DT), clinicopathologic prognostic factors, and cytometric predictors were examined. METHODS. One hundred fifty-eight patients with the possibility to calculate mammographic tumor DT were selected and the tumors were histologically reexamined and flow cytometric analysis for ploidy and S-phase fraction (SPF) was performed. RESULTS. The tumors were Stage I in 68%, and 45% were detected by mammographic screening. DT ranged from 0.6 months to an indefinite time (median, 9.0 months). Short DT was significantly correlated to large tumor size (P = 0.01) and advanced pathologic tumor stage (P = 0.016), but there was no correlation between DT and histologic grade. Ploidy analysis indicated that there were 57% aneuploid and 7% tetraploid tumors. There was a significant overrepresentation of euploid tumors among tumors smaller than 10 mm (P = 0.02). Ploidy was correlated to histologic grade (P less than 0.001) and DT (P = 0.009). SPF was calculated in 122 cases. SPF correlated significantly with pathologic stage (P = 0.002), tumor size (P = 0.037), histologic grade (P = 0.001), the presence of axillary lymph node metastases (P = 0.046), DT (P = 0.02), and DNA ploidy (P less than 0.001). Compared with interval carcinoma, screening-detected carcinoma showed favorable characteristics concerning size, stage, DT, ploidy, and SPF but not regarding histologic grade and axillary lymph node metastases. CONCLUSIONS. DT shows great variations. Factors related to tumor biology (i.e., DT, DNA ploidy, and SPF) are strongly correlated with one another, but they have no correlation with axillary lymph node metastases. Cancer detected by screening is discovered at an early stage and shows favorable characteristics concerning DT, ploidy, and SPF.     

Mammographic growth rate, DNA ploidy, and S-phase fraction analysis in breast carcinoma. A prognostic evaluation in a screened population.

BACKGROUND. The authors examined prognostic factors in 158 cases of breast carcinoma with known mammographic tumor volume doubling times (DT). METHODS. The tumors were retrospectively reexamined histologically and flow cytometric analysis of DNA ploidy and S-phase fraction (SPF) was performed on archival paraffin-embedded material in each case. Life tables and Cox multivariate analyses were used for statistical evaluation of prognostic factors. RESULTS. In univariate analysis of survival data, clinical and pathologic stage, histologic grade, the presence of axillary lymph node metastases, and SPF were significant prognostic predictors, but mammographic DT and DNA ploidy were not. SPF also contributed prognostic information in the subgroup of carcinoma cases detected by screening. In a Cox multivariate analysis, SPF, the presence of axillary lymph node metastases, and Stage II-III disease (as opposed to Stage I disease) were independent significant predictors of survival. In univariate analyses of distant disease-free survival, clinical and pathologic stage, tumor size, histologic grade, the presence of involved axillary nodes, DT, and SPF all were significant prognostic factors. CONCLUSIONS. SPF, stage, and lymph node status were important prognostic factors in this patient material with predominantly small and node-negative breast carcinomas, whereas DNA ploidy and mammographic DT provided less prognostic information. The prognosis of carcinoma detected during screening did not differ significantly from that of breast carcinoma discovered otherwise in this selected patient group.     

Apoptotic and mitotic indices predict survival rates in lymph node-negative colon carcinomas.

An imbalance between apoptosis and mitosis is believed to underlie colon cancer development and progression. These processes regulate the growth of normal and neoplastic epithelia, and in tumors, may confer prognostic information. To test this hypothesis, we determined apoptotic and mitotic indices (AI, MI) by morphology in H&E sections of 154 lymph node-negative, sporadic colon carcinomas. The relationship of these indices to genetic (p53 and Bcl-2) and biological features (DNA ploidy and cell kinetics) and patient survival rates was determined. Tumor features were compared in proximal and distal tumors, given postulated differences in their pathogenesis. Bcl-2 and p53 proteins were examined using immunohistochemistry and DNA ploidy and proliferative indices (PIs) by flow cytometry. Tumor features were dichotomized for analysis of relapse-free survival and overall survival (OS) rates using a Cox proportional hazards model. Median patient follow-up was 8.8 years. The median AI and MI were 1.2% (0-7.6) and 0.40% (0-1.8), respectively, and did not differ by tumor site. AI correlated with histological grade (P = 0.03); MI correlated with PI (P = 0.02) and inversely with Bcl-2 in distal tumors (P = 0.02). p53 and Bcl-2 expression were detected in 52 and 53% of tumors, respectively. Distal tumor site was associated with aneuploidy (P = 0.001), p53 (P = 0.001), and PI > 15% (P = 0.002). In a univariate analysis, colon cancers with high MIs (>0.5%) had a poor prognosis (P = 0.04). Bcl-2 overexpression (>20% + tumor cells) was associated with more favorable OS (P = 0.04). The association of ploidy and PI with outcome was of borderline significance for all tumors; however, diploidy predicted better survival in proximal cancers. In distal cancers, low AIs (< or = 0.25%) and high MIs (>0.5%) were adverse prognostic markers. After adjustment for other variables, an increased MI predicted shorter OS with a hazard ratio (HR) for death of 2.70; 95% confidence interval (CI) was 1.23-5.91 (P = 0.01). Expression of Bcl-2 was associated with more favorable OS (HR, 0.46; 95% CI, 0.21-1.0; P = 0.06). In proximal cancers, Bcl-2 expression was the most important predictor of OS (HR, 0.17; 95% CI, 0.03-0.85; P = 0.03). In distal tumors, low AIs (HR, 3.33; 95% CI, 1.27-9.09; P = 0.01) and high MIs predicted poor survival. In conclusion, increased mitosis and low or absent Bcl-2 expression are significant risk factors for death in node-negative colon cancers, as are low rates of apoptosis in distal tumors. If validated prospectively, our results may identify patient subsets than can benefit from adjuvant chemotherapy.     

The expression of proliferating cell nuclear antigen in paraffin sections of peripheral, node-negative non-small cell lung cancer.

Cell proliferation of 40 peripheral, node-negative non-small cell lung cancers (NSCLC) treated with surgery alone was investigated by immunohistochemical analysis with the monoclonal antibody (MoAb) PC10, which recognizes a proliferating cell nuclear antigen (PCNA) in formalin-fixed and paraffin-embedded material. Results were correlated with DNA ploidy and S-phase fraction (SPF) analyzed by DNA flow cytometric study. Mitotic count (MC) was analyzed by light microscopic study and histopathologic features. PCNA immunoreactivity was seen in all samples and confined to the nuclei of cancer, but not to the surrounding, tumor-negative cells; its frequency ranged from 0-70% (median, 15%), and tumors expressed either a low (0-25%, n = 25) or intermediate (26-75%, n = 15) proliferative activity. There was no relationship between PCNA immunoreactivity and tumor stage or among size, histologic type, and mitotic count (MC). Tumors with intratumoral blood vessel invasion (BVI) showed a significantly higher (P less than 0.005) PCNA immunoreactivity than BVI-negative tumors. PCNA scores were significantly higher (P less than 0.005) in DNA aneuploid (n = 22) than in DNA diploid (n = 18) tumors and correlated significantly with the SPF of DNA aneuploid tumors (r = 0.825, P less than 0.0001), but not with diploid tumors (r = 0.002, P = 0.9). Intermediate proliferating tumors had a significantly higher (P less than 0.01) MC than their counterparts. In univariate analysis, significant predictors of survival were tumor classification (T1 versus T2), tumor size (less than or equal to 2.6 cm versus more than 2.6 cm), BVI (BVI-negative versus BVI-positive), MC (less than or equal to 8 versus more than 8), and PCNA immunoreactivity (low versus intermediate). DNA ploidy and SPF did not influence survival significantly. Only PCNA immunoreactivity retained its independent level of significance (P = 0.02) by multivariate analysis. It was concluded that PCNA immunostaining is a simple and clinically useful method for estimating cell proliferation in formalin-fixed, paraffin-embedded tissue of resected peripheral, node-negative NSCLC.     

Ki-67 immunostaining in node-negative stage I/II breast carcinoma. Significant correlation with prognosis.

Prognostic predictors for node-negative breast carcinoma have not been clearly established. Immunostaining with Ki-67 antibody was performed on frozen sections of histologically proved node-negative breast carcinomas from 42 patients to examine its prognostic value and its association with other clinicopathologic and biochemical parameters, i.e., patient age and tumor size, histologic type, nuclear grade, mitotic rate, presence of vascular or lymphatic invasion, DNA ploidy, percentage of cells in S-phase, estrogen content, and c-erbB-2 amplification. Thirty-seven of the 42 tumors showed immunoreactivity with Ki-67 antibody in 1% to 55% of the tumor cells. A strongly significant correlation was observed between Ki-67 staining percentage and, respectively, nuclear grade, age, and mitotic rate. Nuclear grade 1 (the most anaplastic) tumors showed a significantly higher median percentage of cells stained (median, 14; range, 3 to 40) compared with nuclear grade 3 tumors (median, 0.5; range, 0 to 8). Thirteen patients developed recurrence; six of them died of disease. On univariate analysis, both 5-year disease-free and overall survivals were strongly associated with percentage of cells stained with Ki-67 antibody. Our results suggest that Ki-67 immunostaining correlates well with nuclear grade and clinical outcome in node-negative breast carcinoma. Because of small sample size analyzed in this study we were unable to do multivariate analysis. Therefore, further studies with larger number of cases are needed to determine whether tumor proliferative activity determined by Ki-67 immunostaining is an independent prognostic parameter or it merely reflects histopathologic features such as nuclear grade or mitotic activity.     

DNA flow cytometric analysis and prognosis of axillary lymph node-negative breast carcinoma.

METHODS. The prognostic significance of flow cytometric analysis in patients with node-negative invasive breast carcinoma was evaluated in a retrospective series of 158 patients with a minimum follow-up study of 9 years. RESULTS. The ploidy status could be assessed in 147 specimens (93%), and the proliferative phase or S-phase fraction (SPF) could be assessed in 136 tumors (86%); 70 tumors (48%) were diploid, 49 tumors (33%) were aneuploid, and 28 tumors (19%) were tetraploid. Ploidy status and SPF were correlated significantly with tumor size, histologic grade, nuclear grade, and mitotic rate. By itself, ploidy was not a statistically significant prognostic factor, although all of the patients with multiploid and hypertetraploid tumors had recurrence of disease. The SPF was related significantly to recurrence of disease (P = 0.04). However, when multivariate analysis of various histopathologic variables was performed, SPF ceased to be a significant prognostic determinant, whereas peritumoral lymphovascular invasion was the most important variable. The combination of tumor size and flow cytometric parameters permitted stratification into three groups with different prognoses at the 9-year follow-up review (P less than 0.001). In the low-risk group (diploid tumors less than or equal to 2 cm in diameter with a low SPF or small tetraploid tumors), the recurrence rate was 12%. In the intermediate-risk group (diploid tumors greater than 2 cm in diameter with a low SPF or aneuploid tumors with a low SPF), the recurrence rate was 21%. In the high-risk group (diploid or aneuploid tumors with a high SPF or large tetraploid tumors), the recurrence rate was 49%. The high-risk group status remained a significant variable in the Cox proportional hazards multivariate analysis model. CONCLUSIONS. These results indicate that flow cytometry in breast carcinoma contributes useful but limited prognostic information and stress the importance of using multiple prognostic factors to improve prognostication and optimize patient management.     

New prognostic factors associated with long-term survival in node-negative breast cancer patients

Background  This study was undertaken to determine the absolute and relative value of angiogenesis, proliferating cell nuclear antigen (PCNA) and conventional prognostic factors in predicting relapse-free survival (RFS) and overall survival (OS) rates associated with long-term survival in Japanese patients with node-negative breast cancer. Patients and Methods  Two hundred patients with histological node-negative breast cancer were studied. We investigated nine clinicopathological factors, including angiogenesis, PCNA using permanent-section immunohistochemistry, clinical tumor size, histological grade (HG), tumor necrosis, lymphatic vessel invasion (LVI), histological extension, histological classification, and infiltrating growth (INF), followed for a median of 10 years (range, 1 to 20). Results  Twenty-one patients (10.5%) had recurrence and 15 patients (7.5%) died of breast cancer. Univariate analysis showed that PCNA, clinical tumor size, HG, angiogenesis, and LVI were significantly predictive of 20-year RFS or OS. Tumor necrosis was significantly predictive of OS, not of RFS. Multivariate analysis showed that clinical tumor size (P=0.0003), angiogenesis (P=0.0003), PCNA (P= 0.0064), and HG (P=0.0401) were significant independent prognostic factors for RFS. PCNA (P< 0.0001) and clinical tumor size (P=0.0112) were significant independent prognostic factors for OS, while angiogenesis was a borderline significant factor. Conclusion  PCNA and angiogenesis were important new prognostic factors in node-negative breast cancer patients.